Peroxiredoxin 5 regulates osteogenic differentiation through interaction with hnRNPK during bone regeneration
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Evaluation Summary:
The research presented in this manuscript is focused on testing the role of peroxiredoxin (Prdx5) and heterogeneous nuclear ribonucleoprotein K (hnRNPK) in bone biology and osteoporosis. Using cell-base and animal models, as well as various experimental methodologies the authors demonstrated that Prdx5 is upregulated during osteogenesis but suppressed during osteoclastogenesis. This novel function Prdx5 was found to be associated with binding and regulation of hnRNPK which controls the expression of genes involved in osteoclastogenesis, such as osteocalcin (Ocn).
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Peroxiredoxin 5 (Prdx5) is involved in pathophysiological regulation via the stress-induced cellular response. However, its function in the bone remains largely unknown. Here, we show that Prdx5 is involved in osteoclast and osteoblast differentiation, resulting in osteoporotic phenotypes in Prdx5 knockout ( Prdx5 Ko ) male mice. To investigate the function of Prdx5 in the bone, osteoblasts were analyzed through immunoprecipitation (IP) and liquid chromatography combined with tandem mass spectrometry (LC–MS/MS) methods, while osteoclasts were analyzed through RNA-sequencing. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as a potential binding partner of Prdx5 during osteoblast differentiation in vitro. Prdx5 acts as a negative regulator of hnRNPK-mediated osteocalcin ( Bglap ) expression. In addition, transcriptomic analysis revealed that in vitro differentiated osteoclasts from the bone marrow-derived macrophages of Prdx5 Ko mice showed enhanced expression of several osteoclast-related genes. These findings indicate that Prdx5 might contribute to the maintenance of bone homeostasis by regulating osteoblast differentiation. This study proposes a new function of Prdx5 in bone remodeling that may be used in developing therapeutic strategies for bone diseases.
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Author Response
.Reviewer #1 (Public Review):
- It is important to emphasize that the osteoporotic phenotypes were only demonstrated in males, but not in female mice. The observed phenotypes were not hormone-dependent, as no significant differences in examined bone parameters were observed between wild type andPrdx5KO female mice in an ovariectomy-induced osteoporosis model. However, women over 50 have a four times higher rate of osteoporosis compared with men, and the role of testosterone in the development of osteoporosis in Prdx5KO mice should be investigated. It is known that the osteoporosis is increased in men with low level of testosterone.
Thanks for your comments regarding osteoporosis phenotypes in Prdx5 KO males and their relation with testosterone levels. Based on your suggestion, we re-examined testosterone levels in the …
Author Response
.Reviewer #1 (Public Review):
- It is important to emphasize that the osteoporotic phenotypes were only demonstrated in males, but not in female mice. The observed phenotypes were not hormone-dependent, as no significant differences in examined bone parameters were observed between wild type andPrdx5KO female mice in an ovariectomy-induced osteoporosis model. However, women over 50 have a four times higher rate of osteoporosis compared with men, and the role of testosterone in the development of osteoporosis in Prdx5KO mice should be investigated. It is known that the osteoporosis is increased in men with low level of testosterone.
Thanks for your comments regarding osteoporosis phenotypes in Prdx5 KO males and their relation with testosterone levels. Based on your suggestion, we re-examined testosterone levels in the serum of male mice and tested the expression levels of the androgen receptor (AR) in the differentiated osteoblasts and osteoclasts of the mice. We have updated the data in Figure 3-figure supplement 2 and included the revised information in the Results (Pages 13-14) and Discussion (Page 34) sections.
- It is misleading for authors to state throughout the manuscript that osteoporotic phenotypes are observed in Prdx5KO mice, while it is only observed in male mice.
We apologize for this oversight. We have modified the text and indicated that all osteoporotic phenotypes were observed in Prdx5 KO male mice.
Reviewer #2 (Public Review):
- While the abstract emphasizes transcriptomic analysis and mass spectrometry, extensive imaging techniques have also been used and should be highlighted to give an overview of results from the performed techniques.
In addition, make it clear that it is proteomics-based mass spectrometry, since I was only able to confirm that after seeing Figure 5.
Thanks for your helpful suggestions. We have modified the Abstract based on your suggestions.
- Line 46-53: I would add more details of how balanced bone mass looks on average, how much is too much, when should we be concerned about bone mass, and does some amount of stress benefit bone mass?
Thank you for the suggestion. We have modified the Introduction. We wanted to explain that for bone as a supporting organ, general mechanical stress is required for its remodeling, although we agree that it is not some necessary information related to our study and may confuse the readers.
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Evaluation Summary:
The research presented in this manuscript is focused on testing the role of peroxiredoxin (Prdx5) and heterogeneous nuclear ribonucleoprotein K (hnRNPK) in bone biology and osteoporosis. Using cell-base and animal models, as well as various experimental methodologies the authors demonstrated that Prdx5 is upregulated during osteogenesis but suppressed during osteoclastogenesis. This novel function Prdx5 was found to be associated with binding and regulation of hnRNPK which controls the expression of genes involved in osteoclastogenesis, such as osteocalcin (Ocn).
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This study reveals for the first time the involvement of Prdx5 in the differentiation, of osteoclast and osteoblast, which are key cellular components of bone remodelling. This novel function of Prdx5 was shown to be mediated by specific binding and regulating the function of hnRNPK, which acts as a transcription factor in bone remodelling. Moreover, Prdx5 was shown to be colocalized with hnRNPK during osteoblast differentiation. In support of this findings, the authors also reported that mice deficient for Prdx5 expression (Prdx6KO) showed osteoporosis-like phenotypes, which was associated with reduced osteoblast and increased osteoclast differentiation.
It is important to emphasise that the osteoporotic phenotypes were only demonstrated in males, but not in female mice. The observed phenotypes were not …
Reviewer #1 (Public Review):
This study reveals for the first time the involvement of Prdx5 in the differentiation, of osteoclast and osteoblast, which are key cellular components of bone remodelling. This novel function of Prdx5 was shown to be mediated by specific binding and regulating the function of hnRNPK, which acts as a transcription factor in bone remodelling. Moreover, Prdx5 was shown to be colocalized with hnRNPK during osteoblast differentiation. In support of this findings, the authors also reported that mice deficient for Prdx5 expression (Prdx6KO) showed osteoporosis-like phenotypes, which was associated with reduced osteoblast and increased osteoclast differentiation.
It is important to emphasise that the osteoporotic phenotypes were only demonstrated in males, but not in female mice. The observed phenotypes were not hormone-dependent, as no significant differences in examined bone parameters were observed between wild type and Prdx5KO female mice in an ovariectomy-induced osteoporosis model. However, women over 50 have a four times higher rate of osteoporosis compared with men, and the role of testosterone in the development of osteoporosis in Prdx5KO mice should be investigated. It is known that the osteoporosis is increased in men with low level of testosterone.
It is misleading for authors to state throughout the manuscript that osteoporotic phenotypes are observed in Prdx5KO mice, while it is only observed in male mice.
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Reviewer #2 (Public Review):
This paper examines Peroxiredoxin 5 (Prdx5) and its role in bone differentiation during osteoclast and osteoblast differentiation, opening up therapeutic targets for bone-related diseases.
Abstract:
While the abstract emphasizes transcriptomic analysis and mass spectrometry, extensive imaging techniques have also been used and should be highlighted to give an overview of results from the performed techniques.
In addition, make it clear that it is proteomics-based mass spectrometry, since I was only able to confirm that after seeing Figure 5.
Line 46-53: I would add more details of how balanced bone mass looks on average, how much is too much, when should we be concerned about bone mass, and does some amount of stress benefit bone mass?
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